In general, fossil fuels typified by petroleum and light oil are used as fuels for automobiles. Those fossil fuels, especially, light oil used for diesel automobiles, contain a large amount of nitrogen compound and sulfur compound, so that a large amount of gas such as CO2, NOx, SOx is exhausted from automobiles such as diesel automobiles. Since these exhaust gases cause global warming and environmental pollution, reduction of the exhaust amount is an issue to be solved urgently.
As an alternative fuel to fossil fuels such as light oil, there are high expectations in so-called biodiesel fuel, which uses oils and fate produced by naturally-occurring plants, animals, fishes, or microorganisms. Among these oils and fats, those used for food producing are often dumped into the environment and cause environmental problems. Therefore, expectations in biodiesel fuel made from a waste oil are particularly high in view of prevention of air pollution and effective utilization of a waste oil.
Fatty acid esters obtained by transesterification between an oil and fat and a lower alcohol are preferably used as biodiesel fuels. A variety of researches have been conducted on an enzyme-catalytic method using a lipase, as one method for producing a fatty acid ester (International Publications WO 01/088668 and WO 00/12748). This production method has many advantages, for example, in that aftertreatment of by-product glycerin is easy, in that mild reaction conditions can be applied, and in that a free fatty acid in a starting material can be esterified (H. Fukuda at al., Journal of Bioscience and Bioengineering, 2001, Vol. 92, pp. 405-416).
Regarding production of a fatty acid ester using a lipase, researches have been intensively conducted on a batch-type transesterification in which an enzyme, an oil and fat, and a lower alcohol are agitated and mixed in a screw cap bottle or a reaction tank (Y. Shimada at al., Journal of the American Oil Chemists' Society, 1999, Vol. 76, pp. 789-793, and E. Y. Park et al., Bioresource Technology, 2008, Vol. 99, No. 8, pp. 8180-8185). In this method, it is necessary to be careful of physical damage to the enzyme due to agitation of the reaction mixture. Furthermore, in order to collect a product after the reaction, it is necessary to perform a procedure that separates a product, an enzyme, and a by-product into layers after stopping agitation and allowing the reaction mixture to stand.
On the other hand, there is a report on production of a fatty acid ester using a packed-bed reactor of a tube filled with a lipase through which an oil and fat and a lower alcohol pass (Y. Watanabe et al., Journal of the American Oil Chemists' Society, 2000, Vol. 77, pp. 855-860, and K. Nie et al., Journal of Molecular Catalysis B: Enzymatic, 2006, Vol. 43, pp. 142-147). In this case, the enzyme is fixed in the tube, and, thus, the degree of physical damage to the enzyme is low, and the operation can be performed for a long period of time. Moreover, since the reactor can be filled with a large amount of enzyme, the method has a feature that there is a significant increase in the amount of target substance produced per reactor unit volume and per reaction time. In researches using a packed-bed reactor, typical examples of which are shown by Y. Watanabe et al. (Journal of the American Oil Chemists' Society, 2000, Vol. 77, pp. 855-860) and K. Nie et al. (Journal of Molecular Catalysis B: Enzymatic, 2006, Vol. 48, pp. 142-147), a method is applied in which an oil and fat and a lower alcohol are supplied from an upper portion of a reaction tube, a reaction mixture that has flown out of a lower portion thereof is temporarily allowed to stand so as to be separated into layers, and then a fatty acid ester in an upper layer (also containing unreacted oil and fat) is collected.
In general, a lower alcohol inhibits the activity of a lipase, and thus it is necessary to strictly control the ratio of lower alcohol contained in the reaction mixture. Furthermore, since the solubility of a lower alcohol in an oil and fat is extremely low, it is necessary to keep a uniformly dispersed state such that a droplet of the alcohol is not formed in the oil and fat (Y. Shimada at al., Journal of Molecular Catalysis B: Enzymatic, 2002, Vol. 17, pp. 188-142). There is also a method for reducing the alcohol inhibition by dissolving the reaction mixture in a hydrophobic organic solvent, such as hexane. However, according to the method the collection of the product is difficult, and the production process is complicated.
Glycerin forms as a by-product during a procedure for producing a fatty acid ester. A certain amount of the glycerin accumulated forms a layer around the enzyme. Since this layer of glycerin is hydrophilic, it greatly affects the contact efficiency between unreacted oil and fat and the enzyme. Moreover, part of a lower alcohol remaining in the reaction procedure is dispersed in the glycerin layer, and locally increases the concentration of alcohol near the enzyme, so that a lowered enzymatic activity is caused (Y. Watanabe et al., Journal of the American Oil Chemists' Society, 2000, Vol. 77, pp. 855-860). Conventionally, there is a report on an attempt to remove glycerin by dialysis or by using an organic solvent such as isopropanol (K. B. Bako et al., Biocatalysis and Biotransformation, 2002, Vol. 20, pp. 487-489, and Y. Xu et al., Biocatalysis and Biotranformation, 2004, Vol. 22, pp. 45-48). However, there is a demand for a method for more easily removing glycerin, in view of industrialization of the process.
One aspect is that a method for continuously collecting a product for a long period of time while supplying starting materials is desirable, and use of a packed-bed reactor is advantageous for industrial production of a biodiesel fuel using a lipase. However, another aspect is that it is necessary to be careful of the possibility that a desirable yield of fatty acid ester may not be obtained depending on the supply of an oil and fat and a lower alcohol to a reaction tube and the removal efficiency of by-product glycerin as described above. Therefore, it is necessary to establish a method for continuously producing a biodiesel fuel simultaneously considering these two aspects.